Flow cytometry and cytological studies reveal consecutive rounds of strict partial genome duplications and endomitosis in cellular nuclei from vegetative organs of seeded Dactylorhiza majalis (subfamily Orchidoideae)

Abstract During the analysis of suspensions of PI-stained nuclei from stem, adventitious roots, and leaves of in vitro germinated juvenile D. majalis plants, a range of one to three consecutive rounds of strict partial genome duplications could be discovered. Repeated cycles of under-replications were found again when axenically grown material was moved from solid nutrient media to potting soil, where it further maturated within and outside the laboratory. On average, 83–84% of the 2C DNA content participated in partial genome duplication processes while the remaining 16–17% were untouched. Different frequency distributions were detected for the affected nuclei, depending on the individual, the plant organ, and the ontogenetic stage. The above data were in line with the signs of endomitosis, as shown by acetocarmine-stained smear preparations from the lower epidermis of leaves and from adventitious roots sampled at some distance above the meristematic tip. Endomitosis is one known pathway of endoreplications, which leads to an increased DNA content in non-dividing somatic nuclei. Using a collection of young anthers in the microspore stage, where the regular haplophasic number of 1 n = 2 x = 40 chromosomes could be counted, foliar cells of the related vegetative plant body were considered to contain the whole set of 2 n = 4 x = 80 chromosomes that contributed to a mean mass of 14.71 pg DNA per nucleus at the 2C genome level. The diverse biological source materials and investigated ontogenetic stages as well as the different nuclear DNA amount calculations assessed in this study are of instructional value to avoid erroneous estimations of the present nuclear DNA amount per weight using flow cytometry. This is important with respect to the orchid genus Dactylorhiza , for example, when trying to achieve a full understanding of genome size variation in established polyploids and when searching for nascent polyploids in the future.